Download Impaired mesenchymal cell function in Gata4 mutant mice

Developmental Biology 301 (2007) 602 – 614
www.elsevier.com/locate/ydbio
Genomes & Developmental Control
Impaired mesenchymal cell function in Gata4 mutant mice leads to
diaphragmatic hernias and primary lung defects
Patrick Y. Jay a,b , Malgorzata Bielinska a , Jonathan M. Erlich a , Susanna Mannisto d ,
William T. Pu e , Markku Heikinheimo a,d , David B. Wilson a,c,⁎
a
Department of Pediatrics, Washington University and St. Louis Children’s Hospital, St. Louis, MO 63110, USA
Department of Genetics, Washington University and St. Louis Children’s Hospital, St. Louis, MO 63110, USA
c
Department of Molecular Biology and Pharmacology, Washington University and St. Louis Children’s Hospital, St. Louis, MO 63110, USA
Program for Developmental and Reproductive Biology, Biomedicum Helsinki and Children’s Hospital, University of Helsinki, 00290 Helsinki, Finland
e
Departments of Cardiology, Pediatrics, and Genetics, Children’s Hospital Boston and Harvard Medical School, Boston, MA 02115, USA
b
d
Received for publication 21 May 2006; revised 8 September 2006; accepted 29 September 2006
Available online 5 October 2006
Abstract
Congenital diaphragmatic hernia (CDH) is an often fatal birth defect that is commonly associated with pulmonary hypoplasia and cardiac
malformations. Some investigators hypothesize that this constellation of defects results from genetic or environmental triggers that disrupt
mesenchymal cell function in not only the primordial diaphragm but also the thoracic organs. The alternative hypothesis is that the displacement of
the abdominal viscera in the chest secondarily perturbs the development of the heart and lungs. Recently, loss-of-function mutations in the gene
encoding FOG-2, a transcriptional co-regulator, have been linked to CDH and pulmonary hypoplasia in humans and mice. Here we show that
mutagenesis of the gene for GATA-4, a transcription factor known to functionally interact with FOG-2, predisposes inbred mice to a similar set of
birth defects. Analysis of wild-type mouse embryos demonstrated co-expression of Gata4 and Fog2 in mesenchymal cells of the developing
diaphragm, lungs, and heart. A significant fraction of C57Bl/6 mice heterozygous for a Gata4 deletion mutation died within 1 day of birth.
Developmental defects in the heterozygotes included midline diaphragmatic hernias, dilated distal airways, and cardiac malformations.
Heterozygotes had any combination of these defects or none. In chimeric mice, Gata4−/− cells retained the capacity to contribute to cells in the
diaphragmatic central tendon and lung mesenchyme, indicating that GATA-4 is not required for differentiation of these lineages. We conclude that
GATA-4, like its co-regulator FOG-2, is required for proper mesenchymal cell function in the developing diaphragm, lungs, and heart.
© 2006 Elsevier Inc. All rights reserved.
Keywords: Birth defect; Diaphragm; Pulmonary hypoplasia; Transcription factor
Introduction
Congenital diaphragmatic hernia (CDH) is a severe developmental anomaly that affects 1 per 3000 live births and
accounts for 1–2% of infant mortality (Pober et al., 2005;
Colvin et al., 2005; Yang et al., 2006). The hallmark of the
disorder is a defect in the muscular or tendinous portion of the
diaphragm. CDH is thought to result from abnormal embryonic
development of the diaphragmatic substratum, but the mole⁎ Corresponding author. Department of Pediatrics, Box 8208, Washington
University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110, USA.
Fax: +1 314 286 2892.
E-mail address: [email protected] (D.B. Wilson).
0012-1606/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.ydbio.2006.09.050
cular pathogenesis of this disorder is poorly understood (Greer
et al., 2000b; Babiuk and Greer, 2002).
Primary defects in lung morphogenesis and cardiovascular
malformations often accompany CDH in animal models and
patients (Migliazza et al., 1999; Losty et al., 1999; Graziano,
2005). This association has given rise to the mesenchymal hit
hypothesis, which posits that genetic or environmental triggers
of CDH disrupt the function of mesenchymal cells in not only
the primordial diaphragm but also the developing lungs and
heart (Keijzer et al., 2000). A corollary, the smooth muscle
hypothesis, proposes that disruption of mesenchymal progenitors of smooth muscle in the pulmonary vasculature and airways
leads to pulmonary hypertension, airway hyperreactivity, and
other pulmonary complications commonly encountered in pa-
P.Y. Jay et al. / Developmental Biology 301 (2007) 602–614
tients with CDH (Jesudason, 2006). Consequently, a major goal
of CDH research is to identify genes critical for early mesenchymal cell function in the diaphragm, lungs, and cardiovascular
system.
Recent studies, including analysis of recurring chromosomal
rearrangements in patients with CDH (Lurie, 2003), suggest that
transcription factor haploinsufficiency may cause or contribute
to this disorder. Chromosome 8q22–23 abnormalities have been
associated with CDH (Wilson et al., 1983; Temple et al., 1994;
Howe et al., 1996), and mutation of one of the genes in this
region, FOG2, has been shown to cause diaphragmatic defects
and primary pulmonary hypoplasia in humans and mice
(Ackerman et al., 2005). FOG2 encodes a transcriptional coregulator that is expressed by mesenchymal cells in the
diaphragm, lung, and heart and by somatic cells in the testis
(Svensson et al., 2000; Tevosian et al., 2000; Ketola et al., 2002;
Ackerman et al., 2005). Another recurring chromosomal
abnormality in CDH is microdeletion of 8p23.1 (Pecile et al.,
1990; Faivre et al., 1998; Borys and Taxy, 2004; Shimokawa et
al., 2005; Barber et al., 2005; López et al., 2006). One of the
genes in the critical region of 8p23.1 is GATA4, which encodes
a transcription factor that physically interacts with FOG-2
during morphogenesis of the heart and testis (Crispino et al.,
2001; Tevosian et al., 2002). It is therefore plausible that
GATA4 haploinsufficiency contributes to the pathogenesis of
CDH, particularly in patients with concomitant diaphragm and
heart defects as loss-of-function mutations in GATA4 have been
linked to cardiac malformations in humans (Garg et al., 2003;
Okubo et al., 2004; Nemer et al., 2006) and mice (Kuo et al.,
1997; Molkentin et al., 1997; Crispino et al., 2001; Watt et al.,
2004; Pu et al., 2004; Zeisberg et al., 2005; Xin et al., 2006).
Here we show that C57Bl/6 mice heterozygous for a mutant
allele of Gata4 are predisposed to CDH and primary lung
abnormalities. We propose that GATA-4, working in concert
with FOG-2 or other transcription factors, regulates mesenchymal cell function in the developing diaphragm and lungs. Our
findings support the premise that GATA-4 mutation contributes
to the pathogenesis of CDH and related developmental
anomalies in man.
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XY host blastocysts (Natoli et al., 2004). Only chimeric mice derived from XY
hosts were subjected to further analysis.
Tissue isolation and histological analyses
Late gestation fetuses were harvested, fixed overnight in 4% paraformaldehyde in PBS, and embedded in paraffin for routine histology. In some
cases, pregnant females were injected intraperitoneally with 2 mg bromodeoxyuridine (BrdU) 15 h before embryo harvest. Alternatively, unfixed
fetuses were embedded directly in Tissue-Tek OCT compound (Sakura
Finetek, Torrence, CA) for preparation of cryosections. Thoraces of newborn
pups were isolated by decapitation and transection at the level of the liver.
Skin and soft tissue were removed, and the intact thorax was fixed for
1–2 days in 4% paraformaldehyde + 1% glutaraldehyde in PBS at 4°C.
Diaphragm, heart, and lungs were then dissected and processed for
morphometric and ultrastructural analyses. Paraffin sections (5–6 μm) were
stained with hematoxylin and eosin (H&E) or with Masson's trichrome to
visualize extracellular matrix (ECM).
To detect β-galactosidase (β-gal) expression, frozen tissue sections (10 μm)
were fixed with 0.2% gluteraldehyde for 5 min, permeabilized with 100 mM
potassium phosphate pH 7.4, 0.02% NP-40 and 0.01% sodium deoxycholate for
5–15 min, and then incubated in 0.5 mg/ml X-gal (Promega) with 10 mM K3[Fe
(CN)6], 10 mM K4[Fe(CN)6], 100 mM potassium phosphate pH 7.4, 0.02% NP40, and 0.01% sodium deoxycholate at 30°C overnight (Narita et al., 1997a).
The sections were then counterstained with eosin.
Morphometric analysis
Air space size was estimated from the mean chord length of the airspace
(Ray et al., 1997). Images of H&E-stained lung tissue from postnatal day 1 mice
were acquired at 400× magnification using an Olympus BX60 microscope and a
Zeiss Axiocam digital camera and superimposed on a grid (Ray et al., 1997).
The length of the lines overlying air space air was measured using Scion® Image
software and then averaged to obtain the mean chord length. Fields containing
large airways and vessels were excluded from the chord length measurements.
The number of bronchioles and arteries per mm2 was quantified as described
elsewhere (Mäki et al., 2005).
Electron microscopy
Excised tissue was processed for electron microscopy as described (Narita et
al., 1997a). Briefly, paraformaldehyde/glutaraldehyde-fixed tissue was treated
with OsO4 and then embedded in resin. Sections (1 μm) were cut and stained
with methylene blue for preliminary light microscopic evaluation. Ultrathin
sections were cut, stained with uranyl acetate and lead citrate, and then examined
in a JEM 1010 transmission electron microscope.
Materials and methods
Immunostaining
Experimental mice
Paraformaldehyde-fixed, paraffin-embedded tissue sections were processed
for immunoperoxidase staining using previously described methods (Bielinska et
al., 2005; Jacobsen et al., 2005). The following primary antibodies were employed:
(1) goat anti-mouse GATA-4 IgG (sc-1237, Santa Cruz Biotechnology, Inc., Santa
Cruz, CA), 1:200 dilution; (2) goat anti-mouse FOG-2 (sc-9264, Santa Cruz
Biotechnology, Inc.), 1:100 dilution; (3) rabbit anti-mouse smooth muscle α-actin
(AnaSpec, Inc., San Jose, CA), 1:1000 dilution; (4) rabbit anti-mouse Clara cell
secretory protein CC10 (sc-25555, Santa Cruz Biotechnology, Inc.), 1:100
dilution; (5) goat anti-mouse surfactant protein C (sc-7706, Santa Cruz
Biotechnology, Inc.), 1:200 dilution; (6) goat anti-phosphohistone H3 (sc-12927,
Santa Cruz Biotechnology, Inc.), 1:200 dilution; and (7) mouse anti-BrdU (sc32323, Santa Cruz Biotechnology, Inc.), 1:200 dilution. Secondary antibodies
employed for immunoperoxidase staining were: donkey anti-goat biotinylated IgG
(Jackson Immunoresearch, West Grove, PA) 1:1000 dilution; donkey anti-mouse
biotinylated IgG (Jackson Immunoresearch), 1:2000 dilution; goat anti-rabbit
biotinylated IgG (NEF-813, NEN Life Science, Boston MA), 1:2000 dilution. The
avidin–biotin immunoperoxidase system (Vectastain Elite ABC Kit, Vector
Laboratories, Inc., Burlingame, CA) and diaminobenzidine (Sigma-Aldrich Corp.,
Procedures involving mice were approved by institutional committees for
laboratory animal care and were conducted in accordance with NIH and EU
guidelines for the care and use of experimental animals. C57Bl/6 and Rosa26
(C57Bl/6, Gpi-1b) (Friedrich and Soriano, 1991) mice were purchased from the
Jackson Laboratory (Bar Harbor, ME). Gata4+/Δex2 mice, which harbor a
deletion in exon 2 of the gene, were generated and genotyped as described
elsewhere (Pu et al., 2004; Zeisberg et al., 2005). These mice were backcrossed
with C57Bl/6 mice for a minimum of 7 generations. Nkx2–5+/− mice were
prepared and genotyped as described previously (Tanaka et al., 2002) and were
backcrossed with C57BL/6 mice for a minimum of 12 generations. Chimeric
mice were prepared by injection of XYGata4−/− (CCE, 129/Sv//Ev, Gpi-1c) ES
cells (Soudais et al., 1995; Kuo et al., 1997) into Rosa26 embryos as described
(Narita et al., 1997a). These nullizygous ES cells harbor a neomycin resistance
cassette in exon 2 of the gene. Chimeras were initially identified by GPI-1
isoenzyme analysis of tail tissue (Narita et al., 1997a). XIST RT-PCR analysis of
tail or hind limb tissue was used to distinguish chimeras derived from XX versus
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St. Louis, MO) were used to visualize the bound antibody; slides were then
counterstained with hematoxylin.
electrophoresis (1.2%) in the presence of ethidium bromide demonstrated a
single band of the expected size for each of the PCR primer pairs. Authenticity
of the PCR products was confirmed by direct DNA sequencing.
In situ DNA 3′-end labeling
Apoptosis was quantified in the embryonic diaphragm using an in situ
thymidine deoxyribose-mediated deoxy-UTP nick end labeling (TUNEL) DNA
3′-end labeling kit (Oncor, Gaithersburg, MD). Paraformaldehyde-fixed paraffin
sections were rehydrated through an alcohol series. The permeability of the cell
membranes was increased by incubating the sections in 400 μg proteinase K
(Roche Molecular Biochemicals, Mannheim, Germany) in 200 ml PBS for
15 min. Endogenous peroxidase activity was inhibited by quenching the samples
for 5 min in 5% hydrogen peroxide. DNA fragmentation was identified by
applying terminal transferase enzyme with digoxigenin-labeled nucleotides to
the samples and incubating for 1 h under coverslips. Antidigoxigenin antibody
was used to recognize the digoxigenin-labeled nucleotide chains attached to the
3′-ends of sample DNA. A color reaction was produced with diaminobenzidine
in the presence of 0.03% hydrogen peroxide. The tissue sections were
counterstained with hematoxylin.
RT-PCR
Lung tissue was homogenized in TRIzol (Invitrogen, Carlsbad, CA).
Purified RNA (200 ng) was subjected to RT-PCR using a TITANIUM™ onestep kit (BD Biosciences, Palo Alto, CA), oligo(dT) primers for the reverse
transcriptase reaction, and the PCR primers were as follows: (1) wild-type
Gata4-specific; forward 5′-gaagctgcagcctacggcagt-3′, reverse 5′-gcgcatgtcttcactgctgc-3′, 707 bp; (2) Gata4Δ-specific; forward 5′-tgtcattcttcgctggagcc-3′,
reverse 5′-gcgcatgtcttcactgctgc-3′, 761 bp; Gata6; forward 5′-gcaatgcatgcggcttctac-3′, reverse 5′-ctcttggtagcaccagctca-3′, 554 bp (Johnsen et al.,
2006). Gapdh primers are published elsewhere (Xin et al., 2006). Agarose gel
Results
Gata4 is expressed in early mesenchymal cells of the
diaphragm, lungs, and great vessels
We delineated the developmental expression of GATA-4
protein and mRNA in the diaphragm, lungs, and cardiovascular
system of wild-type mice. Between embryonic day 11.5 (E11.5)
and E15.5, intense GATA-4 immunoreactivity was evident in
tissues critical for diaphragm formation (Greer et al., 2000b),
including the septum transversum (and its derivative the central
tendon), pleuroperitoneal folds (PPF), and esophageal mesenchyme (Figs. 1A, B). During this same period of development,
GATA-4 was observed in mesodermal derivatives in the lung
(mesothelium, submesothelial mesenchyme, and to a lesser
extent subepithelial mesenchyme; Figs. 1A, D). This expression
was most pronounced in the middle and accessory lobes of the
right lung. GATA-4 was also observed in hepatic sinusoidal
cells (Fig. 1B) and cardiac cell types, including cardiomyocytes,
valvuloseptal tissue, and epicardial cells (Fig. 1A and data not
shown). In each of these tissues, the expression pattern of Gata4
overlapped with that of Fog2 (Figs. 1C, E).
Fig. 1. Immunohistochemical staining of diaphragm and lung in the embryonic and newborn mouse. Paraformaldehyde-fixed, paraffin-embedded tissue sections from
either E12.5 (A–E) or P1 (F–I) wild-type mice were subjected to immunoperoxidase staining for GATA-4 (A, B, D, F, G, H), FOG-2 (C, E), or α-smooth muscle actin
(α-SMA) (I) and then counterstained with hematoxylin. Nuclear GATA-4 antigen was seen in tissues that fuse to form the diaphragm, including esophageal
mesenchyme (A, arrowhead), pleuroperitoneal folds, and the central tendon, a derivative of the septum transversum. GATA-4 was also detected in cardiomyocytes,
hepatic sinusoidal cells, pulmonary mesenchymal cells (A, arrows), and mesothelial cells of the visceral pleura. At E12.5, GATA-4 co-localized with FOG-2 in
mesenchymal cells in the central tendon (B, C) and in the terminal acinar buds of the right middle and accessory lobes of the lung (D, E). At P1, some GATA-4
expression persisted in cells of the central tendon (F), especially cells lining the surface of the tendon, but little GATA-4 was evident in the muscular diaphragm (G). In
the newborn lung, GATA-4 was observed in vascular smooth muscle cells (red arrows) but not airway smooth muscle cells (yellow arrows) (H). Control staining with
α-SMA documented expression in both vascular and airway smooth muscle (I). Abbreviations: ct, central tendon of the diaphragm; dv, ductus venosus; e, esophagus;
ht, heart; ivc, inferior vena cava; li, liver; lu, lung; ppf, pleuroperitoneal fold; s, sinusoidal cell of liver; vp, visceral pleura. Scale bars, 75 μm (A–E), 25 μm (F–I).
P.Y. Jay et al. / Developmental Biology 301 (2007) 602–614
Expression of GATA-4 in the diaphragm and lungs decreased at later stages of development. By postnatal day 1
(P1), only ∼50% of the cells in the central tendon exhibited
GATA-4 immunoreactivity (principally those lining the
surface of the tendon; Fig. 1F), and the vast majority of
cells in the muscular diaphragm lacked expression of this
factor (Fig. 1G). In the lungs of E18.5–P1 mice, GATA-4
mRNA and protein were observed in the walls of large and
medium arteries (Figs. 1H; 2B, E) and in mesothelial cells
(Fig. 2H), but there was little or no expression of Gata4 in
airway epithelial cells (Fig. 1H), respiratory smooth muscle
(Figs. 1H, I), small arteries (Fig. 2E), or other pulmonary
interstitial cells. By comparison, transcripts encoding GATA6, a transcription factor implicated in differentiation of pulmonary epithelium and vascular smooth muscle (Keijzer et
al., 2001; Yang et al., 2002), were detected in distal airway
epithelial cells (Figs. 2C, F, I), large pulmonary arteries (Fig.
2C), small pulmonary arteries (Fig. 2F), and mesothelial
cells.
Thus, GATA-4, like its co-regulator FOG-2, is expressed in
mesenchymal and mesothelial cells of the diaphragm and lungs
during the embryonic development of these organs. This pattern
of expression implies that GATA-4 could play a functional role
in organogenesis of not only the heart (Crispino et al., 2001;
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Watt et al., 2004; Pu et al., 2004; Zeisberg et al., 2005; Xin et al.,
2006) but also the diaphragm and lungs.
Neonatal deaths among C57Bl/6 mice heterozygous for
a Gata4 deletion mutation
Gata4 null mice derived from either heterozygote matings
(Kuo et al., 1997; Molkentin et al., 1997) or through diploid/
tetraploid complementation of Gata4−/− ES cells (Narita et al.,
1997b; Watt et al., 2004) die by E10, so insights into diaphragm
or lung development cannot be ascertained from these animals.
We found that ∼ 40% of C57Bl/6 mice harboring a single copy
of a Gata4 mutant allele died within 1 day of birth, irrespective
of gender (Fig. 3). This allele, termed Gata4Δex2 (Pu et al.,
2004), has a large deletion in exon 2 of the gene, which removes
the normal translation start site and N-terminal activation
domain. The allele was generated using Cre-lox technology and
lacks a neomycin resistance cassette or other plasmid sequences
that might affect expression of neighboring genes.
Among newborn offspring of Gata4+/Δex2 matings, the ratio
of wild-type mice to heterozygotes did not deviate from the
expected Mendelian ratio of 1:2. Thus, Gata4 heterozygosity
did not appear to be associated with a high rate of intrauterine
demise on this genetic background. Pathological analysis of
Fig. 2. Expression of GATA-4 and GATA-4 transcripts in late gestation mouse lung. Serial cryosections from a wild-type mouse (E18.5) were stained with H&E
(A, D, G) or subjected to in situ hybridization for GATA-4 (B, E, H) or GATA-6 (C, F, I). Dark-field views are shown in panels B, C, E, F, H, and I. The arrows in
panels D–F highlight respiratory epithelium. The arrowheads in panels G–I indicate the pleural surface of the right lung. Note that GATA-4 is expressed in the walls of
large arteries (B) and in mesothelium (H, arrowheads) but not small arteries (E) or respiratory epithelium. In contrast, GATA-6 is expressed in both large (C) and small
arteries (F) as well as distal airway epithelium (F, I) and mesothelium (I). Abbreviations: br, bronchus; la, large artery; sa, small artery. Scale bars, 50 μm.
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P.Y. Jay et al. / Developmental Biology 301 (2007) 602–614
Fig. 3. Neonatal mortality among C57Bl/6 Gata4 heterozygotes. Gata4+/Δex2
males were mated with either Gata4+/Δex2 or wild-type females and survival of the
resultant offspring monitored. A total of 42 offspring from 8 matings were
genotyped. Kaplan–Meier survival curves were prepared using Statview®
software. Gata4+/Δex2 mice had an inferior overall survival rate due to early
neonatal deaths (P < 0.05, Mantel–Cox Rank test). Gender did not impact survival
(data not shown). Neonatal deaths among Gata4+/+ mice were presumed to reflect
maternal neglect as autopsy of these animals showed no evidence of birth defects.
Gata4+/Δex2 mice, including healthy-appearing adults, revealed
developmental anomalies in the diaphragm, lungs, and heart.
The prevalence of these birth defects in 14 consecutive heterozygotes is summarized in Fig. 4. Overall, ∼ 70% of the heterozygotes in this series had a defect in at least one of these organs.
We conclude that on the C57Bl/6 background Gata4Δex2
heterozygosity is associated with congenital anomalies of the
diaphragm, lungs, and heart, which may occur in isolation or in
combination. This paper focuses on anomalies of the diaphragm
and lungs in these mice. Cardiac anomalies, including a range of
potentially fatal lesions in the newborn period, such as ventricular septal and atrioventricular canal defects, will be discussed
in a separate report.
Expression of normal and variant Gata4 transcripts in the
heterozygotes
To confirm a reduction in Gata4 expression in the
heterozygotes, we performed semi-quantitative RT-PCR on
samples of embryonic lung using PCR primers that were
specific for the wild-type allele. The level of wild-type Gata4
mRNA in the lungs of E14.5 Gata4+/Δex2 mice was 51% that of
littermate controls (P < 0.05, two-tailed t-test), whereas the level
of Gata6 mRNA in the lungs of heterozygotes was similar to
that of controls (Figs. 5A, B).
Using RT-PCR, we found that the Gata4Δex2 allele was
transcribed in embryonic (E13.5–E14.5) but not newborn lung
(Figs. 5A, C). In two other tissues, the heart and liver,
expression of Gata4Δex2 mRNA was evident in both embryonic
and newborn mice (Fig. 5C). If translated using an alternative
ATG initiation codon (Arceci et al., 1993), this transcript could
give rise to a truncated protein that lacks the N-terminal
activation domain but retains both zinc fingers. Such a protein
might act as a hypomorph or in a dominant-negative manner.
Therefore, the phenotype of the heterozygotes may reflect
insufficient amounts of wild-type GATA-4, the production of a
mutant protein, or both.
Diaphragmatic defects in Gata4+/Δex2 mice
Diaphragmatic hernias were observed in neonatal and adult
Gata4 heterozygotes. The hernias arose in the ventral midline
and allowed abdominal viscera (liver and gallbladder) to
protrude into the thoracic cavity (Fig. 6A). Each hernia was
covered by a sac that was continuous with the diaphragm.
Portions of the hernia sac near but not adherent to the liver
contained a mixture of hepatocytes and connective tissue cells
[Fig. 6A, proximal hernia sac (phs)], whereas the more
peripheral aspects of the sac were composed exclusively of
connective tissue [Fig. 6A, distal hernia sac (dhs)]. Slit3−/− mice
display similar hernia sac histopathology, which indicates a
failure of the liver and central tendon to separate during
development, a process normally completed before birth (Yuan
et al., 2003; Liu et al., 2003). In one newborn heterozygote,
herniation was associated with ischemic changes in the median
lobe of the liver, which were visible on both gross inspection
(Fig. 6B) and histological sections (Fig. 6B, inset). In all
newborn wild-type mice, the liver and central tendon were
separated by peritoneal reflections, and trichrome staining
revealed well-organized collagen bundles in the central tendon
(Fig. 6C, left). On the other hand, some Gata4+/Δex2 mice
Fig. 4. Phenotypic variability among Gata4+/Δex2 mice. Fourteen successive heterozygotes, ranging in age from P1 to P100, were analyzed. A shaded box indicates the
presence of a developmental defect in the diaphragm (e.g., herniation or fusion of diaphragm to the liver), lungs (e.g., distal airway dilatation or delayed airway
epithelial cell differentiation), or heart. We observed incomplete penetrance and variable expressivity of Gata4 heterozygosity on this genetic background.
Abbreviations: a, secundum atrial septal defect (ASD); av, atrioventricular canal (AV-canal); v, ventricular septal defect (VSD).
P.Y. Jay et al. / Developmental Biology 301 (2007) 602–614
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2004). TUNEL staining of E13.5 embryos showed increased
apoptosis in the amuscular diaphragm of Gata4+/Δex2 mice
compared to littermate controls (Fig. 7B vs. A). To determine
whether Gata4Δex2 heterozygosity was associated with de-
Fig. 5. Expression of normal and variant Gata4 transcripts in the heterozygotes. (A) RT-PCR was performed using RNA isolated from E14.5 lungs of
Gata4+/+ (+/+) and Gata4+/Δex2 (+/Δ) mice. Duplicates are shown. The PCR
primers were specific for wild-type GATA4, Gata4Δex2 (GATA4Δ), GATA6,
or GAPDH. (B) Relative mRNA expression normalized to GAPDH in wildtype (filled bars; n = 3) and Gata4+/Δex2 (open bars, n = 3) embryos is shown.
The asterisk indicates a significant downregulation of Gata4 mRNA in the
heterozygotes (P < 0.05, two-tailed t-test). (C) RT-PCR for the GATA4Δ transcript was performed using RNA from lungs, heart, or liver of Gata4+/+ (+/+)
and Gata4+/Δex2 (+/Δ) mice (ages E13.5 and P1).
exhibited abnormal fusion of the central tendon to the liver
surface, and trichrome staining revealed disorganized collagen
bundles (Fig. 6C, right).
Among Gata4 heterozygotes, the overall incidence of
diaphragmatic defects (frank herniation or aberrant fusion of
central tendon to liver documented on histological sections) was
6/21 (29%). Overt herniation was seen in 3/21 (14%). No
diaphragmatic defects were observed on gross or histological
inspection of Gata4+/+ littermate controls (n = 15; P < 0.05,
two-population proportions test). Nor were diaphragmatic
hernias observed on gross inspection of a larger number of
unrelated wild-type C57Bl/6 mice (n = 54; ages P116–P766;
P < 0.05) or C57Bl/6 mice heterozygous deficient in another
cardiac transcription factor, Nkx2–5 (n = 87; ages P140–P373;
P < 0.01).
Reduced expression of Gata4 has been associated with
increased apoptosis and impaired cellular proliferation in the
heart and other tissues (Suzuki and Evans, 2004; Aries et al.,
Fig. 6. Diaphragmatic defects in Gata4+/Δex2 mice. (A) Diagram of a
diaphragmatic hernia in an adult Gata4+/Δex2 mouse. The photographs show
H&E-stained sections from different regions in the herniated tissue. Both liver
(median lobe) and gallbladder were found in the hernia sac. The portion of the
hernia sac adjacent to liver contained a mixture of hepatocytes and connective
tissue, indicating incomplete separation of the diaphragm and liver. The distal
portion of the hernia sac was composed exclusively of connective tissue and
was continuous with the amuscular diaphragm. (B) Stereomicroscopic view of
the liver through the translucent diaphragm of a P1 Gata4+/Δex2 mouse. The
dashed line shows the boundary between the central tendon and muscular
diaphragm. The left lateral aspect of the median lobe of the liver is ischemic
due to incarceration in a retrosternal diaphragmatic hernia (arrow). The inset
shows an H&E-stained section of the ischemic liver tissue. Note the extensive
parenchymal necrosis. (C) Left panel. Trichrome-stained diaphragm from a P1
Gata4+/+ (+/+) mouse, illustrating separation of the central tendon from the
liver. Right panel. Trichrome-stained diaphragm from a P1 Gata4+/Δex2 (+/Δ)
mouse. Note fusion of the diaphragm and liver and the presence of
disorganized fibrils in the central tendon. Abbreviations: ct, central tendon;
gb, gallbladder; hs, hernia sac; il, ischemic liver (median lobe); lv, liver
(normal); phs, proximal hernia sac; pr, peritoneal recess; r, rib; st, sternum.
Scale bars, 1 mm (B), 50 μm (C).
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Fig. 7. Impact of Gata4 heterozygosity on apoptosis and cellular proliferation in the embryonic diaphragm. Paraformaldehyde-fixed, paraffin-embedded tissue
sections from BrdU-labeled E13.5 Gata4+/+ (A, C, E) or Gata4+/Δex2 (B, D, F) mice were subjected to TUNEL staining (A, B) or immunoperoxidase staining for
BrdU (C, D) or phosphohistone H3 (E, F). The frequencies of TUNEL-, BrdU-, and phosphohistone H3-positive cells in Gata4+/Δex2 (open bars; n = 4) mice versus
littermate controls (filled bars; n = 3) are shown in the bar graphs. Note the statistically significant increase in apoptosis in the diaphragm of Gata4+/Δex2 embryos.
Scale bars, 30 μm.
creased cellular proliferation, we performed BrdU labeling. At
E13.5, there was no difference in BrdU incorporation in the
amuscular diaphragm in Gata4+/ex2 versus wild-type embryos
(Fig. 7D vs. C). Similar results were obtained when proliferation
was measured by staining for the M-phase specific marker
phosphohistone H3 (Fig. 7F vs. E).
We conclude that heterozygosity for the Gata4 deletion
mutation predisposes C57Bl/6 mice to develop diaphragmatic
hernias in the ventral midline. Gata4Δex2 heterozygosity does
not impair cell proliferation but leads to increased apoptosis of
cells in the amuscular diaphragm, which may contribute to the
development of diaphragmatic hernias.
Pulmonary abnormalities in Gata4+/Δex2 mice
On gross inspection, the lungs of Gata4+/Δex2 mice (ages
E16.5–P60) resembled control lungs in overall dimensions and
lobation pattern. Internally, however, dilated distal airways and
patches of thickened mesenchyme were observed in the lungs of
some newborn heterozygotes (Fig. 8A vs. B). Airway dilatation
was most pronounced in the accessory and middle lobes of the
right lung (i.e., sites where mesenchymal Gata4 expression is
especially pronounced). Measurement of mean chord length
(Ray et al., 1997), a gauge of airway diameter, confirmed a
significant increase in saccule size in the Gata4+/Δex2 vs.
control mice (Table 1). The overall incidence of distal airway
dilatation among Gata4 heterozygotes (ages P1–P8) was 7/21
(33%). No dilatation was observed on histological inspection of
Gata4+/+ controls (n = 10; P < 0.05). The number of proximal
airways was similar in Gata4+/Δex2 and Gata4+/+ mice, but
there was a statistically insignificant decrease in the number of
arteries/mm 2 in the heterozygotes (Table 1).
To determine the impact of Gata4Δex2 heterozygosity on the
differentiation of endodermal and mesodermal lineages, we
performed electron microscopy and immunostaining on lungs
from newborn Gata4+/Δex2 mice and wild-type controls.
Ultrastructural analysis showed that proximal airways in
Gata4+/Δex2 lungs were lined by normal appearing Clara cells
and ciliated cells (Fig. 8C), and the dilated distal airways were
lined by cells with the typical features of mature type 1 and type
P.Y. Jay et al. / Developmental Biology 301 (2007) 602–614
609
Fig. 8. Abnormalities in the lungs of Gata4+/Δex2 mice. (A, B) Paraformaldehyde-fixed, paraffin-embedded sections of lung from a P1 Gata4+/+ (A) or Gata4+/Δex2
(B) mouse were stained with H&E. Note the enlarged distal airspaces in the Gata4+/Δex2 lung tissue. Neither a heart malformation nor diaphragm defect was evident in
this Gata4+/Δex2 mouse, suggesting that airway dilatation was not a secondary phenomenon. (C, D) Transmission electron microscopy of proximal (C) and distal (D)
airway epithelium from Gata4+/Δex2 mice. The proximal airway epithelium contains ciliated cells and Clara cells, the latter recognizable by the typical dome-shaped
apical cytoplasmic protrusions into the airway lumen. The lumen of distal airway is dilated; cells with the hallmark features of type 1 pneumocytes, type 2
pneumocytes, and endothelial cells are evident. (E–H) Paraformaldehyde-fixed, paraffin-embedded sections of lung from a P1 Gata4+/+ mouse (E, F) or P1 Gata4+/Δex2
(G, H) mouse were subjected to immunoperoxidase staining for CCSP (E, G) or SPC (F, H). Note the reduced expression of both the proximal and distal epithelial
markers in the Gata4+/Δex2 lung tissue. Lung specimens in panels C, D, G, and H were from the same heterozygote. Abbreviations: cil, ciliated airway cell; cla, Clara
cell; p1, da, dilated (distal) airspace; en, endothelial cell; fb, fibroblast; p1, type 1 pneumocyte; p2, type 2 pneumocyte; rbc, red blood cell. Scale bars, (A, B) 300 μm,
(C, D), 3 μm, (E–H) 30 μm.
2 pneumocytes (Fig. 8D). However, immunohistochemistry
revealed reduced or delayed expression of both Clara cell
secretory protein (CCSP; Fig. 8E vs. G) and the type 2
pneumocyte marker, surfactant protein C (SPC; Fig. 8F vs. H),
in some P1 mice. This impaired expression of airway markers
was evident in only 2/20 (10%) of Gata4+/Δex2 mice and was
limited to those with the most severe distal airway dilatation.
Expression of smooth muscle α-actin (α-SMA) in pulmonary arteries and airway smooth muscle was similar in wild-type
mice (Fig. 9A) and Gata4 heterozygotes (Fig. 9C). On the other
hand, ectopic expression of α-SMA was evident in mesothelial
and submesothelial cells lining the right middle and accessory
lobes of the heterozygotes (Fig. 9B vs. D). This aberrant αTable 1
Morphometric analysis of wild-type and Gata4+/Δex2 lungs at P1
Mean chord length (μm)
# bronchioles/mm2
# arteries/mm2
⁎ P < 0.05, Student's t-test.
Wild-type (n = 6)
Gata4+/Δex2 (n = 6)
20 ± 2.8
8.8 + 2.1
16.9 + 1.7
29 ± 3.1⁎
8.2 + 1.6
14.7 + 2.4
SMA expression, which was observed in 10% of heterozygotes,
might reflect trans-differentiation of mesothelial cells to
myofibroblasts or de-repression of the α-SMA gene. Intriguingly, ectopic α-SMA expression has been observed (Shikama
et al., 2003) in the pleura of mice bearing a mutant of p300, a
known cofactor of GATA-4 (Dai and Markham, 2001).
In summary, Gata4+/Δex2 mice are predisposed to distal
airway dilatation and specific alterations in pulmonary gene
expression, affecting both endoderm and mesoderm derivatives.
The presence of ectopic α-SMA expression in Gata4+/Δex2
lungs suggests that GATA-4 might directly or indirectly
function as a repressor of certain genes. Since some Gata4
heterozygotes exhibited lung defects in the absence of CDH
(Fig. 5), we infer that the pulmonary abnormalities are not
merely secondary to diaphragmatic defects.
Absence of GATA-4 does not block mesenchymal cell
differentiation in the diaphragm or lung
As noted above, Gata4−/− embryos exhibit severe malformations on both pure (129 or C57Bl/6) and mixed (129:
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P.Y. Jay et al. / Developmental Biology 301 (2007) 602–614
Fig. 9. Ectopic expression of α-SMA in the lungs of Gata4+/Δex2 mice. Sections of paraformaldehyde-fixed, paraffin-embedded lungs from P1 Gata4+/+ (A, B) or
Gata4+/Δex2 (C, D) mice were subjected to immunoperoxidase staining for α-smooth muscle actin (α-SMA) followed by hematoxylin counterstaining. Proximal
airways are shown in panels A and C, while distal airways and pleura are shown in panels B and D. In both Gata4+/+ and Gata4+/Δex2 lungs, α-SMA is seen in airway
smooth muscle (yellow arrowheads) and vascular smooth muscle (black or white arrows). Robust expression of α-SMA was observed in mesothelial cells and
subjacent mesenchymal cells in Gata4+/Δex2 but not Gata4+/+ lungs (compare red arrows in panel B vs. panel D). Abbreviations: da, dilated (distal) airway; pa,
proximal airway. Scale bars, 50 μm.
C67Bl/6) genetic backgrounds (Kuo et al., 1997; Molkentin et
al., 1997; Narita et al., 1997b; Watt et al., 2004). We therefore
wondered whether Gata4−/− progenitors might exhibit a block
in early mesenchymal cell differentiation regardless of genetic
background. To test this possibility, we generated chimeric
mouse embryos by injecting Gata4−/− ES cells (129/Sv//Ev)
into Rosa26 (C57BL/6) embryos. Both copies of the Gata4
gene in these ES cells bear a neomycin resistance cassette,
which abrogates mRNA expression (Soudais et al., 1995; Kuo
et al., 1997). Rosa26 mice bear a ubiquitously expressed β-gal
transgene that facilitates lineage tracing (Friedrich and Soriano,
1991).
Nullizygous ES cells retained the capacity to contribute to
the central tendon of E14.5 chimeras, as demonstrated by X-gal
staining (Fig. 10A). In situ hybridization of adjacent tissue
sections verified that these central tendon cells lacked
expression of Gata4 (Fig. 10B) but maintained expression of
another mesenchymal marker, Gata6 (Fig. 10C). Similarly,
Gata4−/− ES cells contributed to mesenchymal cells and
mesothelial cells in the lungs of E15.5 chimeras (data not
shown).
We did not observe diaphragmatic hernias or overt lung
anomalies among chimeric embryos (n = 7, 25–60% chimerism by GPI isozyme analysis). High percentage Gata4−/−
chimeras do not survive to term (Narita et al., 1997a,b; Watt et
al., 2004), precluding an analysis of the impact of GATA-4
absence on postnatal diaphragm or lung development. Based
on analysis of fetal chimeras, we conclude that GATA-4 is not
essential for differentiation of mesenchymal cells in the
diaphragm or lungs.
Discussion
Despite advances in antenatal diagnosis, surgical techniques,
and medical management, the morbidity and mortality associated with CDH remain substantial (Colvin et al., 2005; Smith
et al., 2005). Approximately half of the patients die in the
neonatal period, and survivors often have longstanding
cardiopulmonary dysfunction (West and Wilson, 2005).
Although theoretically promising, fetal surgery has not been
shown to improve outcome for infants with this condition.
Consequently, a major focus of ongoing CDH research is on
disease prevention, which mandates a better understanding of
its pathogenesis.
As with other developmental defects, our ability to
characterize the molecular basis of CDH is dependent on
animal models, and the most widely used is exposure of fetal
rodents to nitrofen (Greer et al., 2000a). Diaphragmatic hernias
have been observed in several genetically engineered mouse
strains, including those with germline homozygous mutations
of Wt1 (Kreidberg et al., 1993; Moore et al., 1998, 1999), Slit3
(Yuan et al., 2003; Liu et al., 2003), the Slit receptor Robo1
(Xian et al., 2001), Fog2 (Ackerman et al., 2005), or Lox (Mäki
et al., 2002; Hornstra et al., 2003; Mäki et al., 2005). More
recently, conditional mutagenesis of Coup-TFII in the foregut
mesentery and PPF has been shown to cause CDH in mice (You
et al., 2005). Supporting the mesenchymal hit hypothesis, some
of these mouse strains predisposed to CDH have attendant
defects in the lungs or heart.
We describe here a novel mouse model of CDH based on
heterozygosity of a Gata4 deletion mutation. Like other genes
P.Y. Jay et al. / Developmental Biology 301 (2007) 602–614
Fig. 10. Gata4−/− cells retain the capacity to differentiate into mesenchymal
cells in the diaphragm. Adjacent cryosections through an E14.5 Gata4−/− ↔
Rosa26 chimera were stained with X-gal (A) or subjected to in situ
hybridization for Gata4 (B) and Gata6 (C). Dark-field views are shown in
panels B and C. Note the presence of β-gal negative (Gata4−/−) cells in the
central tendon (A, arrowheads). These cells lack Gata4 mRNA (B, arrowheads)
but express abundant Gata6 mRNA (C, arrowheads). Control experiments (not
shown) documented β-gal and Gata4 mRNA in the central tendon of agematched non-chimeric mice. Abbreviations: ht, heart; liver, lv. Scale bars,
100 μm.
implicated in CDH, Gata4 is expressed in the embryonic
structures that fuse to form the diaphragm. Gata4+/Δex2 mice
develop midline diaphragmatic hernias resembling those seen in
Slit3−/− mice (Yuan et al., 2003; Liu et al., 2003). Increased
apoptosis in the diaphragmatic substratum of the heterozygotes
(Fig. 7) may compromise tensile strength and contribute to
hernia formation. The type of diaphragmatic hernia seen in the
Gata4+/Δex2 mouse differs from the posterolateral (Bochdalek)
hernia typically seen in patients with CDH. These differences in
anatomic pathology might be species-specific. Modeling
studies suggest that diaphragm morphogenesis in rodents is
dominated by the PPF and may not be strictly applicable to man
(Fisher and Bodenstein, 2006).
In addition to CHD, Gata4+/Δex2 mice are predisposed to
primary pulmonary developmental defects, including dilated
distal airspaces, impaired (or delayed) expression of airway
epithelial markers, and ectopic expression of α-SMA in
mesothelium. Proper lung development requires coordinated
interactions between endoderm-derived airway epithelial cells
and mesodermal derivatives (mesothelium, submesothelial
611
mesenchyme, subepithelial mesenchyme, etc.) (Shannon and
Hyatt, 2004; Borok et al., 2006; White et al., 2006). Since
pulmonary expression of GATA-4 is restricted to mesodermal
derivatives, the airway defects in Gata4 heterozygotes are
presumably the indirect effects of perturbed signaling from
mesenchyme or mesothelium to endoderm. Similar abnormalities in airway development have been observed in mice deficient in certain ECM proteins, including elastin (Wendel et al.,
2000), lysyl oxidase (Mäki et al., 2005), fibulin-4 (McLaughlin
et al., 2006), fibulin-5 (Yanagisawa et al., 2002), laminin α5
(Nguyen et al., 2005), and laminin γ2 (Nguyen et al., 2006), and
in mice deficient in the transcription factors Foxf1 (Mahlapuu et
al., 2001; Kalinichenko et al., 2004), FOG-2 (Ackerman et al.,
2005), and p300 (Shikama et al., 2003). In several of these
cases, lung abnormalities were more pronounced in the middle
and accessory lobes of the right lung.
Phenotypic similarities between Fog2−/− (Ackerman et al.,
2005) and Gata4+/Δex2 mice suggest that the main activity
affected by FOG-2 deficiency in the diaphragm and lungs is
GATA-4 rather than another GATA factor. Supporting this
premise, a recent abstract (Ackerman et al., 2006) reported
primary defects in lung development in mice harboring a Gata4
missense mutation that disrupts GATA4–FOG interactions; the
pulmonary phenotype of these Gata4 missense mutants
resembled that of Fog2−/− mice. Traditionally, GATA-6, a
factor expressed in pulmonary epithelium, has been viewed as
the sole member of the GATA family required for lung
morphogenesis (Keijzer et al., 2001; Yang et al., 2002); it is
now apparent that GATA-4 also influences pulmonary development through its expression in mesodermal cells.
Our observations connecting murine Gata4 mutations to
CDH and related developmental anomalies are consistent with
other lines of evidence. First, microdeletions spanning the
human GATA4 gene on 8p23.1 have been linked to cases of
CHD, including cases associated with cardiac malformations
typical of GATA4 haploinsufficiency (Faivre et al., 1998;
Devriendt et al., 1999; Shimokawa et al., 2005; Barber et al.,
2005). In fact, most reported cases of CDH in del(8)(p22.1pter) had an accompanying cardiac malformation, especially
atrioventricular canal defects (Lurie, 2003). Indeed, it has
been suggested that deletion 8p23.1 should be considered
whenever the combination of diaphragmatic hernia and AV
canal is encountered (Faivre et al., 1998). Second, the
expression and activity of GATA-4 are influenced by retinoids
(Arceci et al., 1993; Kostetskii et al., 1999; Clabby et al.,
2003; Ghatpande et al., 2006), and perturbed retinoid
metabolism has been linked to malformations of the
diaphragm, lungs, and heart (reviewed in Greer et al., 2003;
Gallot et al., 2005; Montedonico et al., 2006). Ultimate proof
that GATA-4 is involved in the pathogenesis of human CDH
awaits the identification of patients with diaphragmatic hernias
and small deletions or point mutations within the GATA4
gene. Given the established role of GATA-4 in heart and testis
development, such patients with CDH might be predicted to
have concomitant cardiac malformations (Pehlivan et al.,
1999; Garg et al., 2003; Okubo et al., 2004) or male-to-female
sex reversal (Meacham et al., 1991; Maaswinkel-Mooij and
612
P.Y. Jay et al. / Developmental Biology 301 (2007) 602–614
Stokvis-Brantsma, 1992; Manouvrier-Hanu et al., 2000;
Killeen et al., 2002; Kent et al., 2004).
Our findings, coupled with those of other investigators
(Kreidberg et al., 1993; Ackerman et al., 2005; You et al., 2005),
suggest that proper development of the diaphragm requires the
concerted action of several transcription factors, including
GATA-4, FOG-2, COUP-TFII and WT-1. We speculate that
these factors regulate a set of genes critical for mesenchymal
cell function in the diaphragmatic substratum as well as the
lungs and heart. Preliminary analysis of Fog2−/− mouse
embryos suggests that the hepatocyte growth factor gene may
be one of the targets (Ackerman et al., 2005). Whatever the
target genes are, we note that they may be abnormally expressed
in some but not all affected animals or even within the cells or
tissues of the same animal, as shown, for example, by the
random ectopic expression of α-SMA in regions of the pleura of
some Gata4 heterozygotes. The molecular mechanism of
stochastic gene expression is unknown but could underlie the
ostensibly random combination of diaphragm, lung, and heart
defects in mutant animals and human patients.
Earlier studies of Gata4 heterozygous deficient mice failed
to detect CDH or any other obvious birth defect (Kuo et al.,
1997; Molkentin et al., 1997; Watt et al., 2004), although Gata4
heterozygotes have been shown to have increased sensitivity to
doxorubicin-induced cardiotoxicity (Aries et al., 2004). Differences in genetic background probably impact the development
of birth defects in Gata4 heterozygotes since previous studies
employed mixed backgrounds (Kuo et al., 1997; Molkentin et
al., 1997) rather than a C57Bl/6 “congenic” strain. The Gata4
loss-of-function allele used here (Gata4Δex2) differs from that
used in other studies (Kuo et al., 1997; Molkentin et al., 1997;
Watt et al., 2004; Aries et al., 2004), so allele-specific effects
might account for some of the findings.
We postulate that epigenetic influences and stochastic events
contribute to the phenotypic variability in the Gata4Δex2
heterozygotes on the C57Bl/6 background (Cook et al., 1998;
Magee et al., 2003). This incomplete penetrance may be
exploited for future mechanistic studies; C57Bl/6 Gata4+/Δex2
mice may serve as a “sensitized” strain that is useful for probing
the effects of vitamin A deficiency, teratogens, and modifier
genes on the development of CDH and related anomalies.
Acknowledgments
We thank Karen Hutton in the DDRCC Histology Core for
her assistance. We thank Brian Hackett for helpful discussions.
This research was supported by NIH HL61006 and DK52574,
MOD FY02-203, Mallinckrodt Foundation, Finnish Pediatric
Research Foundation, and the Juselius Foundation. PYJ is a
Scholar of the Child Health Research Center of Excellence in
Developmental Biology at Washington University School of
Medicine (K12-HD001487).
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